Functional analysis and gene expression profiling of extracellular cathepsin Z in red sea bream, Pagrus major.

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major, and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N-glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida: E. piscicida and Streptococcus iniae: S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ, and the leukocytes of red sea bream also induced apoptosis and viability reduction.

Characterization, expression profiling and functional characterization of cathepsin Z (CTSZ) in turbot (Scophthalmus maximus L.).

Cathepsin Z (CTSZ) is a lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. In this study, we reported the identification of SmCTSZ, a CTSZ homolog from turbot (Scophthalmus maximus L.). SmCTSZ was 317 residues in length and contains a Pept-C1 domain. In multiple species comparison, SmCTSZ shared 65-93% overall sequence identities with the CTSZ counterparts from human, rat, and several fish species. In the phylogenetic analysis, SmCTSZ showed the closest relationship to Cynoglossus semilaevis. The syntenic analysis revealed the similar neighboring genes of CTSZ across all the selected species, which suggested the synteny encompassing CTSZ region during vertebrate evolution. Subsequently, SmCTSZ was constitutively expressed in various tissues, with the lowest and highest levels in brain and intestine respectively. In addition, SmCTSZ was significantly up-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSZ showed strong binding ability to all the examined microbial ligands, and the agglutination effect to different bacteria. Taken together, these results indicated SmCTSZ could play important roles in mucosal immune response in the event of bacterial infection in teleost. However, the knowledge of CTSZ are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes.

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.